3.3.- Flux Balance Analysis of the reduced model
Parsimonious Flux Balance Analysis approach (p-FBA) was performed using Optflux 3.4 Software (Rocha et al., 2010), a user-friendly computational tool for metabolic engineering applications. p-FBA is a variant of flux balance analysis that minimizes the total material flow to achieve an objective, while maintaining the optimal growth (Lewis et al., 2010). The graphical representation of the p-FBA was generated using Omix Visualization Software (GmbH&Co.KG), as described in Droste et al. (Droste et al., 2011), where only the most significant fluxes for this study were represented.
As usual in FBA with genomic models, the system was under-determined and there existed some degrees of freedom. To find the optimal state, p-FBA uses the optimization of a certain objective function, in this case the maximum value of ATP generation (EF0002). Cytoplasmic malic enzyme was considered to be inactive and NADPH required for biosynthesis could only be regenerated by pentose phosphate pathway, as this degree of freedom can only be solved using more advanced fluxomic techniques (R00216 [0 0]) (Quek et al., 2010). Glutaminolysis was constrained to go in the direction of oxoglutarate generation (R00243mitoc [0 10000]) (McKeehan, 1986). Specific consumption/production rates of experimental metabolites measured were added to the model using the mean of the duplicates ± standard deviation.